PETRI-seq

PETRI-seq is based on SPLiT-seq, but it is optimised for prokaryotic single-cell RNA-seq, such as for bacteria. The method uses random priming during the reverse transcription to capture prokaryotic RNA. The procedures are very similar to those in SPLiT-seq.

The PETRI-seq was published in Nature Microbiology 5, 1192-1201 (2020). There is also an accompanying website. The workflow described in this page is based on the protocol from the website, and the oligo sequences are also taken from the website. For a backup, I copied the protocol (accessed on 26-Jan-2024) and the oligo table (accessed on 26-Jan-2024) into this GitHub.


Adapter and primer sequences:

*Round1 RT Primers (randN): 5'-/5Phos/ GCCAGA[7-bp Round1 barcode]NNNNNN -3'

*Round2 Ligation Oligos: 5'-/5Phos/ GCTTCGC[7-bp Round2 barcode]CCTCCTAC -3'

*Round3 Ligation Oligos: 5'- AGAATACACGACGCTCTTCCGATCT[7-bp UMI][7-bp Round3 barcode]GGTCCTTG -3'

Round 2 barcode linker (SB83): 5'- STCTGGCGTAGGAGGW -3'

Round 3 barcode linker (SB80): 5'- GCGAAGCCAAGGACCW -3'

Round 3 blocking strand 1 (SB81): 5'- GCTTCGCTGCAATCGGACCTCGATTGCA -3'

Round 3 blocking strand 2 (SB82): 5'- WGGTCCTTGGCTTCGC -3'

Round 2 blocking strand 1 (SB84): 5'- GCCAGASACGTTAGGCAGGACCTAACGT -3'

Round 2 blocking strand 2 (SB85): 5'- WCCTCCTACGCCAGAS -3'

Nextera Tn5 binding site (19-bp Mosaic End (ME)): 5'- AGATGTGTATAAGAGACAG -3'

Nextera N/S5xx primer entry point (s5): 5'- TCGTCGGCAGCGTC -3'

Nextera N7xx primer entry point (s7): 5'- GTCTCGTGGGCTCGG -3'

NEB i50x primer (NEB kit E7600): 5'- AATGATACGGCGACCACCGAGATCTACAC[8-bp i5 index]ACACTCTTTCCCTACACGACGCTCTTCCGATCT -3'

Nextera (XT) N7xx Index primer: 5'- CAAGCAGAAGACGGCATACGAGAT[8-bp i7 index]GTCTCGTGGGCTCGG -3'

Illumina TruSeq Read 1 primer: 5'- ACACTCTTTCCCTACACGACGCTCTTCCGATCT -3'

Index 1 sequencing primer (i7): 5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'

Index 2 sequencing primer (i5): 5'- AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT -3'

Illumina Nextera Read 2 primer: 5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'

Illumina P5 adapter: 5'- AATGATACGGCGACCACCGAGATCTACAC -3'

Illumina P7 adapter: 5'- CAAGCAGAAGACGGCATACGAGAT -3'

*NOTE: The first nt of the 7-bp Round1 barcode is C or G, that is S; The last nt of 7-bp Round2&3 barcode is A or T, that is W.

Step-by-step library generation

(1) Prepare ligation adapters by annealing barcodes with corresponding linkers (in two different plates):


Anneal Round2 Ligation Oligos with Round 2 barcode linker (SB83):

5'-/5Phos/ GCTTCGC[7-bp Round2 barcode]CCTCCTAC
                                  3'- WGGAGGATGCGGTCTS -5'


Anneal Round3 Ligation Oligos with Round 3 barcode linker (SB80):

5'- AGAATACACGACGCTCTTCCGATCT[7-bp UMI][7-bp Round3 barcode]GGTCCTTG
                                                       3'- WCCAGGAACCGAAGCG -5'

(2) Anneal Round1 RT primer to mRNA and reverse transcription using Maxima H Minus Reverse Transcriptase in situ:


5'-/5Phos/ GCCAGA[7-bp Round1 barcode]NNNNNN--->
                           3'- XXXXXXX...XXXXXXX -5'

(3) Pool cells and re-distribute to annealed Round2 plate for Round2 Barcode Ligation:


5'-/5Phos/ GCTTCGC[7-bp Round2 barcode]CCTCCTACGCCAGA[7-bp Round1 barcode]NNNNNN--->
                                  3'- WGGAGGATGCGGTCTS -5'     3'- XXXXXXX...XXXXXXX -5'

(4) Pool and re-distribute to annealed Round 3 plate for for Round3 Barcode Ligation:


5'- AGAATACACGACGCTCTTCCGATCT[7-bp UMI][7-bp Round3 barcode]GGTCCTTGGCTTCGC[7-bp Round2 barcode]CCTCCTACGCCAGA[7-bp Round1 barcode]NNNNNN--->
                                                       3'- WCCAGGAACCGAAGCG -5'            3'- WGGAGGATGCGGTCTS -5'     3'- XXXXXXX...XXXXXXX -5'

(5) Pool, lyse cells, cDNA purification and 2nd strand synthesis using the NEBNext Second Strand Synthesis system (presumably an RNaseH + DNA pol I based method), and purify the double stranded cDNA:


5'- AGAATACACGACGCTCTTCCGATCT[7-bp UMI][7-bp Round3 barcode]GGTCCTTGGCTTCGC[7-bp Round2 barcode]CCTCCTACGCCAGA[7-bp Round1 barcode]XXX.XXX -3'
3'- TCTTATGTGCTGCGAGAAGGCTAGA[7-bp UMI][7-bp Round3 barcode]CCAGGAACCGAAGCG[7-bp Round2 barcode]GGAGGATGCGGTCT[7-bp Round1 barcode]XXX.XXX -5'

(6) Tagmentation with Illumina Nextera XT Kit:

Tn5 dimer 

Product 1 (s5 at both ends, not amplifiable due to PCR primers used, see the next step):

5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX         CTGTCTCTTATACACATCT
                  TCTACACATATTCTCTGTC         XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGACTGCGACGGCTGCT -5'



Product 2 (s7 at both ends, not amplifiable due to PCR primers used, see the next step):

5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX         CTGTCTCTTATACACATCT
                   TCTACACATATTCTCTGTC         XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'



Product 3 (different s5 and s7 at both ends, not amplifiable, due to PCR primers used, see the next step):

5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGXXXXXXXXXXXX...XXX         CTGTCTCTTATACACATCT
                  TCTACACATATTCTCTGTC         XXX...XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'


Product 4 (s5 at one end, 3' of cDNA at the other end, not amplifiable due to PCR primers used, see the next step):

5'- AGAATACACGACGCTCTTCCGATCT[7-bp UMI][7-bp Round3 barcode]GGTCCTTGGCTTCGC[7-bp Round2 barcode]CCTCCTACGCCAGA[7-bp Round1 barcode]XXX.XXX         CTGTCTCTTATACACATCT -3'
3'- TCTTATGTGCTGCGAGAAGGCTAGA[7-bp UMI][7-bp Round3 barcode]CCAGGAACCGAAGCG[7-bp Round2 barcode]GGAGGATGCGGTCT[7-bp Round1 barcode]XXX.XXXXXXXXXXXXGACAGAGAATATGTGTAGACTGCGACGGCTGCT -5'


Product 5 (s7 at one end, 3' of cDNA at the other end, the only amplifiable fragment, see the next step):

5'- AGAATACACGACGCTCTTCCGATCT[7-bp UMI][7-bp Round3 barcode]GGTCCTTGGCTTCGC[7-bp Round2 barcode]CCTCCTACGCCAGA[7-bp Round1 barcode]XXX.XXX         CTGTCTCTTATACACATCT -3'
3'- TCTTATGTGCTGCGAGAAGGCTAGA[7-bp UMI][7-bp Round3 barcode]CCAGGAACCGAAGCG[7-bp Round2 barcode]GGAGGATGCGGTCT[7-bp Round1 barcode]XXX.XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'

(7) Use NEB i50X and Nextera XT N7xx primers for library amplification:


5'- AATGATACGGCGACCACCGAGATCTACAC[i5]ACACTCTTTCCCTACACGACGCTCTTCCGATCT-------->
                                         5'- AGAATACACGACGCTCTTCCGATCT[7-bp UMI][7-bp Round3 barcode]GGTCCTTGGCTTCGC[7-bp Round2 barcode]CCTCCTACGCCAGA[7-bp Round1 barcode]XXX.XXX         CTGTCTCTTATACACATCT -3'
                                         3'- TCTTATGTGCTGCGAGAAGGCTAGA[7-bp UMI][7-bp Round3 barcode]CCAGGAACCGAAGCG[7-bp Round2 barcode]GGAGGATGCGGTCT[7-bp Round1 barcode]XXX.XXXXXXXXXXXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
                                                                                                                                                                                                          <----GGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'

(8) Final library structure:


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNGGTCCTTGGCTTCGCNNNNNNNCCTCCTACGCCAGANNNNNNNXXX.XXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNTGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGANNNNNNNNNNNNNNCCAGGAACCGAAGCGNNNNNNNGGAGGATGCGGTCTNNNNNNNXXX.XXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
            Illumina P5           8bp i5          TruSeq Read 1            7-bp   7-bp      Round3      7-bp      Round2     7-bp   cDNA          ME              s7         8bp i7       Illumina P7
                                                                           UMI   Round3     linker     Round2     linker    Round1
                                                                                 barcode               barcode              barcode

Library sequencing:

(1) Add Illumina TruSeq Read 1 sequencing primer to sequence the first read (bottom strand as template, UMI and barcode reads, at least 57 cycles):


                                     5'- ACACTCTTTCCCTACACGACGCTCTTCCGATCT-------------------------------------------------------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNTGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGANNNNNNNNNNNNNNCCAGGAACCGAAGCGNNNNNNNGGAGGATGCGGTCTNNNNNNNXXX.XXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(2) Add Index 1 sequencing primer to sequence i7 index (bottom strand as template, 8 cycles):


                                                                                                                                      5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC------->
3'- TTACTATGCCGCTGGTGGCTCTAGATGTGNNNNNNNNTGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGANNNNNNNNNNNNNNCCAGGAACCGAAGCGNNNNNNNGGAGGATGCGGTCTNNNNNNNXXX.XXXGACAGAGAATATGTGTAGAGGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'

(3) Cluster regeneration, add Index 2 sequencing primer to sequence the i5 index (top strand as template, 8 cycles):


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNGGTCCTTGGCTTCGCNNNNNNNCCTCCTACGCCAGANNNNNNNXXX.XXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
                                 <-------TGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA

(4) Add Illumina Read 2 sequencing primer to sequence the second read (top strand as template, cDNA read, 17 cycles):


5'- AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNGGTCCTTGGCTTCGCNNNNNNNCCTCCTACGCCAGANNNNNNNXXX.XXXCTGTCTCTTATACACATCTCCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'
                                                                                                                                   <------GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'